US5429922A - Composition and method for distinguishing virulent and non-virulent toxoplasma infections - Google Patents
Composition and method for distinguishing virulent and non-virulent toxoplasma infections Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
- C07K14/45—Toxoplasma
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56905—Protozoa
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- C07K2319/00—Fusion polypeptide
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- This invention relates to the field of disease diagnosis and also to the fields of genetic engineering and antibody production. More particularly it relates to the identification of virulent and non-virulent strains of Toxoplasma gondii by antibody binding and restriction fragment length polymorphism and to the preparation of compositions, particularly monoclonal antibodies, useful for such identification.
- Toxoplasmosis is caused by the protozoan parasite, Toxoplasma gondii.
- the disease In humans, the disease is traditionally associated with the developing fetus in whom it can cause severe neurological problems manifesting as hydrocephaly, mental retardation and/or blindness [1, 2].
- the disease In healthy adults, the disease is typically mild, producing few if any symptoms.
- the parasite In immunocompromised adults, however, the parasite can cause severe or even fatal disease [3, 4, 5]. The disease also occurs in other mammals and is the leading cause of spontaneous abortion in sheep.
- the parasite itself is extremely widespread and is typically acquired through the ingestion of undercooked meat in which tissue cysts containing the parasite may reside.
- the parasite can be contacted through ingestion of cat feces which contain the product of the complete sexual cycle, the oocyst. Both cyst forms are stable (the oocysts particularly so) and so avoidance of infection is difficult.
- serological studies indicate that about 15% of the population has had contact with the parasite [6].
- this figure can rise to as much as 85% (e.g., in France [7]).
- the incidence of disease in the developing fetus is, notably, not as high as these figures might at first suggest because it appears that the fetuses of women who contract and control the disease prior to pregnancy are generally not at risk [8].
- Diagnosis of congenital infection has in the past relied on serology (reviewed in [14]). This can be done postnatally or, ideally, pre-natally and relies on the relative titers of IgG and IgM (to deduce whether the titers are due to a current infection or legacy of a past infection).
- the factors contributing to the severity of disease in the developing fetus have been poorly understood. The only well-established factor is that the time of initial infection of the mother relative to conception is critical: infection significantly before conception such that an effective immune response has been mounted by the mother, results in little if any disease.
- toxoplasmosis has dramatically increased in a relatively new group of patients who are in some way immunodeficient as a result of post-transplantation therapy [5, 9, 10], neoplastic disease [11, 12, 13] or, most recently, acquired immunodeficiency syndrome (AIDS) [3, 4, 5].
- AIDS acquired immunodeficiency syndrome
- the parasite can cause a disseminated, potentially fatal form of the disease [5].
- AIDS patients with toxoplasmosis typically first present with significant neuropathy (reviewed in [14]). This is due to the fact that one of the tissues most affected by the parasite is the brain wherein massive parasite cysts can be found. Infection is not limited to the brain, however, and tissue cysts can be found throughout the body [11].
- the typical routine for diagnosis includes serology, computed tomography, magnetic resonance imaging and/or brain biopsy [1, 15, 16]. Of these, the only definitive route to diagnosis is the brain biopsy as this enables the direct visualization of the parasite, using immuno-peroxidase staining [17].
- the course of treatment for toxoplasmosis in pregnant individuals is determined by the stage in pregnancy and whether the infection is acute or chronic. If infection is acute, spiromycin may be administered but is of unproven efficacy. More effective drugs such as combined therapy with pyrimethamine and sulfadiazine are not generally used because of their toxicity for the developing fetus. Hence, these latter drugs are employed in only rare cases where infection of the fetus (as opposed to the mother) is directly demonstrated. Such diagnosis has only been done on an experimental basis.
- toxoplasmosis Treatment of toxoplasmosis in non-pregnant individuals is initiated and maintained with a drug regimen involving a combination of the folate antagonists, pyrimethamine and sulfadiazine [1, 14]. If the disease is identified soon enough, treatment is reasonably effective in combatting the acute disease. However, due to poor tolerance of the drugs, especially of the sulfa compounds in AIDS patients, maintenance on the drug therapy is frequently not possible and recrudescence of the infection is often observed (that is, the drug therapy reduces but does not eliminate the parasite infection).
- the present invention provides diagnostic assays based on the discovery of dimorphism in the p30 antigen and gene and the direct association of this dimorphism with the known bimodal virulence pattern. Assays can be based on different antigenic behavior of virulent (referred to here as type I) and avirulent (also referred to as non-virulent; type II) p30 antigens or on the differences in the corresponding genes. There is a one-to-one relationship between the type I and type II antigen/genes and virulence. Specific genetic materials, specific antibodies, and analytical techniques that allow a diagnosis to be made between virulent and avirulent infections are disclosed in the following detailed description and examples.
- FIGS. 1A-1D presents the cDNA nucleic acid sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of the P30 gene from an avirulent strain and the cDNA nucleic acid sequence (SEQ ID NO: 3) and deduced amino acid sequence (SEQ ID NO: 4) of the P30 gene from a virulent strain RI-I.
- the RH sequences are identical to the avirulent sequences except where indicated.
- the underlined sequences at the beginning and the end of the sequences show respectively the oligonucleotides (sense and antisense) used in the PCR cloning. Numbering is from the first nucleotide of the first in-frame ATG codon.
- the p30 protein is glycolipid-anchored, the C-terminal-most ten or more amino acids are almost certainly removed from the primary translation product.
- Toxoplasma gondii isolates from around the world and from a variety of animal hosts fall into one of two distinct classes based on the following criteria:
- the invention allows the extremely rapid determination of whether the strain responsible for any given clinical infection of animals or humans is virulent and thus informs the physician or veterinarian as to the likely outcome of the infection. This information is extremely important when deciding on the most effective course of therapy (i.e., in deciding to use the most effective but most toxic drugs over the less effective but safer alternatives).
- the invention arose in the laboratories of the present inventors, where earlier studies had discovered genetic material encoding specific proteins of the protozoan parasite Toxoplasma gondii for the first time.
- One of these proteins was the p30 antigen. See U.S. application Ser. No. 07/616,693, filed Nov. 20, 1990, which is a continuation of U.S. application Ser. No. 07/166,384, filed Mar. 9, 1988, now abandoned, both of which are herein incorporated by reference.
- the p30 antigen is a major surface antigen (see Kasper et al., J. Imm. (1983) 130:2407-2412) and can be used for the production of vaccines or diagnostic standards (the latter for use in immunoassays for detecting T. gondii infections in general).
- FIG. 1 shows the coding for virulent and non-virulent p30 genes.
- the main DNA and amino acid sequence lines show the avirulent-strain sequence, with virulent-strain sequence differences being indicated on a separate line below the main sequence lines.
- the four sequences (specified and implied, from top to bottom) are referred to herein as SEQ. ID. NO. 1-4, respectively.
- the present invention is directed to any assay that detects the difference between type I and type II p30 antigens or between type I and type II p30 genes.
- assays based on the identification of antibodies in infected sera that show specific binding for one or the other but not both antigen types are also part of the invention. Since there is a one-to-one correlation between either of these differences and virulence, determination of virulence is now much easier than was possible previously using the mouse LD50 assay.
- the solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activate carboxyl, hydroxyl, or aldehyde group.
- homogeneous assay In a second diagnostic configuration, known as a homogeneous assay, antibody binding to an analyte produces some change in the reaction medium which can be directly detected in the medium.
- Known general types of homogeneous assays proposed heretofore include (a) spin-labeled reporters, where antibody binding to the antigen is detected by a change in reported mobility (broadening of the spin splitting peaks), (b) fluorescent reporters, where binding is detected by a change in fluorescence efficiency, (c) enzyme reporters, where antibody binding effects enzyme/substrate interactions, and (d) liposome-bound reporters, where binding leads to liposome lysis and release of encapsulated reporter.
- spin-labeled reporters where antibody binding to the antigen is detected by a change in reported mobility (broadening of the spin splitting peaks)
- fluorescent reporters where binding is detected by a change in fluorescence efficiency
- enzyme reporters where antibody binding effects enzyme/substrate interactions
- liposome-bound reporters where binding leads to
- some peptide fragments based on these sequences and fragments representing minor variations thereof will have the binding activity of the various complete peptides and can be used in competitive binding assays that distinguish type I and type II p30 antigens.
- fragments of the p30 peptide sequence that are capable of being recognized by immunoglobulins specific for the p30 antigen itself can readily be prepared and screened.
- Peptide synthesizers can be used to prepare small polypeptide fragments (e.g., less than 100 amino acids) or techniques of genetic engineering can be used to prepare larger fragments.
- a simple screening procedure that will identify suitable polypeptide fragments consists of preparing monoclonal antibodies to the appropriate p30 antigen, attaching the antibodies to an affinity column, and capturing peptide fragments that are retained by the bound antibody.
- Polyclonal antisera can be used instead of monoclonal antibodies if desired.
- polypeptide of the invention in the form of a fused polypeptide.
- peptides are typically prepared by using the promoter region of a gene known to be expressed in a host and inserting nucleotides that encode all or a major portion of the amino acid sequence of the invention into the genetic sequence for the host protein.
- a fused protein has been prepared with ⁇ -galactosidase.
- Another technique for preparing immunologically active peptide fragments is to synthesize a series of amino acids of from 5-100 amino acids in length (or any intervening length, such as 10, 15, or any other multiple of 2, 3, or 5 in this range) and screen for immunological activity using an antiserum (or monoclonal antibody).
- the fragments would be selected along the entire length of the peptide to optimize cross-reactivity (e.g., a series of peptides 20 amino acids in length and comprising AA 1 -AA 20 AA 5 -AA 25 , AA 10 -AA 30 , etc.).
- the selected fragment would then correspond to particularly useful corresponding nucleotide sequences that could be used to produce large amounts of the peptide for use as described herein.
- Preferred peptides differ at no more than 12, more preferably no more than 5, amino acids in any contiguous group of 20 amino acids. Standard conservative groups of amino acids are shown in parenthesis using the one-letter amino acid code: nonpolar (A,V,L,I,P,M); aromatic (F,T,W); uncharged polar (G,S,T,C,N,Q); acidic (D,E); basic (K,R,H).
- nonpolar A,V,L,I,P,M
- aromatic F,T,W
- uncharged polar G,S,T,C,N,Q
- acidic D,E
- basic K,R,H
- Antibodies specific for p30 antigen are produced by immunizing an appropriate vertebrate host, e.g., rabbit, with purified p30 antigen or polypeptide derivatives of p30 antigen, by themselves or in conjunction with a conventional adjuvant. Usually, two or more immunizations will be involved, and blood or spleen will be harvested a few days after the last injection. For polyclonal antisera, the immunoglobulins can be precipitated, isolated and purified by a variety of standard techniques, including affinity purification using p30 antigen attached to a solid surface, such as a gel or beads in an affinity column.
- the splenocytes normally will be fused with an immortalized lymphocyte, e.g., a myeloid cell line, under selective conditions for hybridoma formation.
- the hybridomas can then be cloned under limiting dilution conditions and their supernatants screened for antibodies having the desired specificity.
- Techniques for producing antibodies are well known in the literature and are exemplified by the publication Antibodies: A Laboratory Manual (1988) eds. Harlow and Lane, Cold Spring Harbor Laboratories Press, and U.S. Pat. Nos. 4,381,292, 4,451,570, and 4,618,577.
- p30 antigen for use as immunogens in the preparation of anti-p30 antibodies can be readily purified from tissue or blood and its components, such as serum and plasma, taken from infected humans or animals and from cells genetically modified to produce p30 antigen or polypeptide derivatives thereof, by affinity chromatography using a monoclonal antibody specific for p30 antigen.
- p30 antigen and polypeptide derivatives thereof can be purified by a variety of other widely known protein purification techniques (either alone or in combination) including immunoprecipitation, gel filtration, ion exchange chromatography, chromatofocusing, isoelectric focusing, selective precipitation, electrophoresis, and the like.
- Derivatives having the desired immunogenicity can be prepared as described above.
- p30 antigen both glycosylated and unglycosylated, or polypeptide derivatives thereof, may be used for producing antibodies, either monoclonal or polyclonal, specific to p30 antigen.
- polypeptide derivatives polypeptides differing in length from natural p30 antigen and containing five or more amino acids from p30 antigen in the same primary order as found in p30 antigen as obtained from a natural source.
- Polypeptide molecules having substantially the same amino acid sequence as p30 antigen but possessing minor amino acid substitutions that do not substantially affect the ability of the p30 antigen polypeptide derivatives to interact with p30 antigen-specific molecules, such as antibodies, are within the definition of p30 antigen.
- Derivatives include glycosylated forms, aggregative conjugates with other p30 antigens molecules and covalent conjugates with unrelated chemical moieties. Covalent derivatives are prepared by linkage of functionalities to groups which are found in the p30 antigen amino acid chain or at the N- or C-terminal residue by means known in the art.
- Monoclonal antivirus particle antibodies or anti-idiotype antibodies can be produced as follows.
- the spleen or lymphocytes from an immunized animal are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art.
- a human lymphocyte donor is selected.
- Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes or a human fusion partner can be used to produce human-human hybridomas.
- EBV Epstein-Barr virus
- Primary in vitro immunization with peptides can also be used in the generation of human monoclonal antibodies.
- Antibodies secreted by the immortalized cells are screened to determine the clones that secrete antibodies of the desired specificity.
- Monoclonal antibodies against p30 have been prepared previously but not screened for the higher specificity required of the present invention. However, the screening technique is straightforward and easily carried out.
- An antibody composition (polyclonal or monoclonal) is prepared using either the type I or type II p30 antigen (from any strain) as an immunogen. After an initial screening to determine that the antibody composition actually binds with the immunogen, negative screening is carried out to select those antibody compositions that do not bind to the other member of the bimodal antigen pair. The resulting antibody compositions are capable of distinguishing between type I and type II p30 antigens and can be used in diagnostic techniques of the invention.
- the target of the analysis is a specific antibody in the blood or other body fluid of an infected human or animal that shows the indicated specific binding for one but not both of the p30 antigen types. No purification, isolation, or handling of the parasite itself is then required.
- Numerous such assays exist (such as the tests now used for the AIDS antibody) and can be adapted to the present invention simply by substituting a type I or type II antigen (or any of the drerivatives or fragments discussed above that have the indicated specificity) for the antigen normally used in the assay.
- a preferred assay would check for positive binding to one antigen type and negative binding to the other, particularly when using entire p30 molecules which have multiple determinants.
- a small fragment is used that is known (by the screening process used to obtain the fragment) to bind only with a type-discriminating antibody
- a single binding assay is preferred.
- a competitive binding assay can be used in which a labelled antibody standard known to discriminate between the two types competes with unknown antibody present in a sample.
- Assays of the invention can also be based on gene differences rather than antigen differences. Any conventional technique can be used. In most cases, amplification of the genetic material in the sample is desirable.
- One method for amplification of target nucleic acids, for later analysis by hybridization or other assays, is known as the polymerase chain reaction or PCR technique.
- the PCR technique can be applied to detecting p30 genes of the invention in suspected samples using oligonucleotide primers spaced apart from each other and based on the genetic sequence set forth herein.
- the primers are complementary to opposite strands of a double stranded DNA molecule and are typically separated by from about 50 to 450 nt or more (usually not more than 2000 nt).
- This method entails preparing the specific oligonucleotide primers and then repeated cycles of target DNA denaturation, primer binding, and extension with a DNA polymerase to obtain DNA fragments of the expected length based on the primer spacing. Extension products generated from one primer serve as additional target sequences for the other primer.
- the degree of amplification of a target sequence is controlled by the number of cycles that are performed and is theoretically calculated by the simple formula 2n where n is the number of cycles. Given that the average efficiency per cycle ranges from about 65% to 85%, 25 cycles produce from 0.3 to 4.8 million copies of the target sequence.
- Oligonucleotide adaptors, probes, and sequencing and PCR primers can be synthesized by the phosphoramidite method with an Applied Biosystems (Foster City, Calif.) model 380A synthesizer, purified by polyacrylamide gel electrophoresis, and desalted on SEP-PAK C 18 cartridges (Waters; Milford, Mass.).
- PCR reactions can be performed according to the supplier of the PCR kit (Perkin/Elmer/Cetus) using the PCR primers described (see description of oligonucleotide synthesis).
- the DNA is amplified, it can be analyzed by a variety of techniques that are used to detect differences between two different DNA sequences, such as restriction fragment length polymorphism (RFLP) analysis and selective hybridization.
- RFLP restriction fragment length polymorphism
- RFLP analysis relies on the ability of restriction endonucleases to generate DNA fragments of different length when two DNA molecules have a difference at the binding/cleavage site of the enzyme.
- DNA obtained from samples is incubated with at least one restriction endonuclease that cleaves either the type I gene or the type II gene but not both.
- Other endonucleases can be used that cleave both types at common sites in order to cut the molecules in convenient lengths for analysis.
- Characteristic oligonucleotide fragments will be generated whose length depends on the type of p30 gene present. Analysis of the fragment lengths (usually on an electrophoresis gel) allows ready determination of which type p30 gene was present in the original sample, and thus ready determination of virulence.
- oligonucleotide probes from the regions of variation of the two nucleotide sequences disclosed herein. Such probes will be relatively short because the differences between the sequences are minor, and long probes may not distinguish between the probes because the binding affinities are too similar. Probes at least 10, preferably at least 14, nucleotides in length, are desirable. Intermediate oligonucleotides up to 50, especially up to 20 or 30, nucleotides in length provide particularly specific and rapid-acting probes. Both RNA and DNA probes can be used. The hybridization conditions (especially temperature and salt concentration) are adjusted as necessary to provide differential hybridization.
- the probes are typically labelled in a detectable manner (e.g., with 32p, 3H, biotin, or avidin) and are incubated with single-stranded DNA or RNA from the organism in which a gene is being sought.
- Hybridization is detected by means of the label after single-stranded and double-stranded (hybridized) DNA (or DNA/RNA) have been separated (typically using nitrocellulose paper).
- Hybridization techniques suitable for use with oligonucleotides are well known.
- oligonucleotide probe refers to both labeled and unlabeled forms.
- nucleotide sequences up to or even greater than 100 bases long can be readily synthesized on an Applied Biosystems Model 380A DNA Synthesizer as evidenced by commercial advertising of the same (e.g., Genetic Engineering News, November/December 1984, p. 3).
- Such oligonucleotides can readily be spliced using, among others, the technique of preparing overlapping complementary sequences (e.g, 1-100 of coding strand, 0-50 and 51-150 of complementary strand, 101-200 of coding strand, etc.) followed by hybridizing and ligating the strands.
- DNA molecules that code for such peptides can readily be determined from standard lists of codons and are likewise contemplated as being equivalent to the DNA sequence of Table 1.
- any discussion in this application of a replacement or other change in a peptide is equally applicable to the corresponding DNA sequence or to the DNA molecule, recombinant vector, or transformed microorganism in which the sequence is located (and vice versa).
- DNA (or corresponding RNA) molecules of the invention can have additional nucleotides preceding or following those that are specifically listed.
- poly A can be added to the 3'-terminal, short (e.g., fewer than 20 nucleotides) sequence can be added to either terminal to provide a terminal sequence corresponding to a restriction endonuclease site, stop codons can follow the peptide sequence to terminate translation, and the like.
- DNA molecules containing a promoter region or other control region upstream from the gene can be produced.
- DNA molecules containing the sequences of the invention will be useful for at least one purpose since all can minimally be fragmented to produce oligonucleotide probes or polymerase chain reaction (PCR) primers and be used in the isolation or detection of DNA from biological sources.
- PCR polymerase chain reaction
- genes and corresponding proteins can be prepared by the totally synthetic techniques discussed above, in preferred embodiments of the invention genetic information is obtained from natural sources and identified as described herein, as previously mentioned.
- the genetic material is first obtained in the form of a gene library, using any of numerous existing techniques. The first of these is to randomly shear genomic DNA and insert this sheared material into expression vectors. If enough recombinants are generated, there is a good probability of having at least one recombinant in the population which is expressing a fusion protein corresponding to the antigen of interest. In practice, for a genome the size of T. gondii (about 7 ⁇ 107 bp), at least 5 ⁇ 106 independent recombinants are needed.
- a clone obtained in the manner described above has been fully sequenced. This sequence was used to isolate other cDNA clones. Together, these sequences can be used to predict the complete protein-coding sequence of the gene as shown in Table 1.
- the primary translation product has a predicted Mr of 36,210 kD. It also has a probable hydrophobic signal peptide at its N-terminus, as expected for a surface antigen. It has one predicted N-glycosylation site (residue 267) consistent with investigations of previous workers which have indicated that the p30 protein may be a glycoprotein. Finally, it has a hydrophobic C-terminus which is not followed by any charged residues. This is apparently diagnostic of a process originally reported in trypanosomes whereby the hydrophobic polypeptide segment is replaced by a glycolipid anchor. Such a process is now known to occur for major surface antigens of Leishmania and Plasmodium as well.
- the gene encoding the p30 antigen can be used for the production of full or modified peptides using standard techniques of manipulating and growing unicellular microorganisms.
- Antigens which are candidates for vaccine development and/or diagnostic reagents will include those recognized by serum from infected patients.
- T. gondii protein can be enhanced by including multiple copies of the gene in a transformed host, by selecting a vector known to reproduce in the host, thereby producing large quantities of protein from exogenous inserted DNA (such as pUC8; ptac12; pIN ompA1, 2, or 3; pOTS; pAS1; or pKK223-3), or by any other known means of enhancing peptide expression.
- exogenous inserted DNA such as pUC8; ptac12; pIN ompA1, 2, or 3; pOTS; pAS1; or pKK223-3
- a T. gondii protein will be expressed when the DNA sequence is functionally inserted into the vector.
- functionally inserted is meant in proper reading frame and orientation, as is well understood by those skilled in the art.
- a gene will be inserted downstream from a promoter and will be followed by a stop codon, although production as a hybrid protein (possibly followed by cleavage) may be used, if desired.
- U.S. Pat. No. 4,419,450 discloses a plasmid useful as a cloning vehicle in recombinant DNA work.
- U.S. Pat. No. 4,362,867 discloses recombinant cDNA construction methods and hybrid nucleotides produced thereby which are useful in cloning processes.
- U.S. Pat. No. 4,403,036 discloses genetic reagents for generating plasmids containing multiple copies of DNA segments.
- U.S. Pat. No. 4,363,877 discloses recombinant DNA transfer vectors.
- U.S. Pat. No. 4,336,336 discloses a fused gene and a method of making the same.
- U.S. Patent 4,349,629 discloses plasmid vectors and the production and use thereof.
- U.S. Pat. No. 4,332,901 discloses a cloning vector useful in recombinant DNA.
- kits for the detection of Toxoplasmosis gondii, where the improvement allows virulent and non-virulent strains to be distinguished.
- kits will comprise a reagent selected from the group consisting of (1) for kits that determine the presence of T.
- gondii by the detection of specific nucleotide sequences (a) restriction endonucleases that cleave either type I or type II p30 gene, but not both; (b) oligonucleotides that hybridize under the reaction conditions provided by the kit with either type I or type II p30 gene, but not both; and (c) antibodies that bind with either type I or type II p30 gene, but not both; and (2) for kits that determine the presence of T. gondii by the detection of a specific antigen binding event, specific binding molecules that bind either type I or type II p30 gene, but not both.
- Other kits can be prepared with other reagents that allow detection according to the general methods discussed above or in the examples below.
- homogeneous when referring to a peptide or DNA sequence, that the primary molecular structure (i.e., the sequence of amino acids or nucleotides) of substantially all molecules present in the composition under consideration is identical.
- the term "substantially” as used in the preceding sentence preferably means at least 95% by weight, more preferably at least 99% by weight, and most preferably at least 99.8% by weight.
- fragments derived from entire molecules of the homogeneous peptide or DNA sequence if present in no more than 5% by weight, preferably 1% by weight, and more preferably 0.2% by weight, is not to be considered in determining homogeneity since the term "homogeneous" relates to the presence of entire molecules (and fragments thereof) have a single defined structure as opposed to mixtures in which several molecules of similar molecular weight are present but which differ in their primary molecular structure.
- isolated refers to pure peptide, DNA, or RNA separated from other peptides, DNAs, or RNAs, respectively, and being found in the presence of (if anything) only a solvent, buffer, ion or other component normally present in a biochemical solution of the same. "Isolated” does not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure substances or as solutions.
- pure as used herein preferably has the same numerical limits as “substantially” immediately above.
- replacement by or replacement does not necessarily refer to any action that must take place but to the peptide that exists when an indicated “replacement” amino acid is present in the same position as the amino acid indicated to be present in a different formula (e.g., when leucine is present at amino acid 3 of p30 instead of valine).
- Salts of any of the peptides described herein will naturally occur when such peptides are present in (or isolated from) aqueous .solutions of various pHs. All salts of peptides having the indicated biological activity are considered to be within the scope of the present invention. Examples include alkali, alkaline earth, and other metal salts of carboxylic acid residues, acid addition salts (e.g., HCl) of amino residues, and zwitter ions formed by reactions between carboxylic acid and amino residues within the same molecule.
- Toxoplasma gondii which is the most commonly used laboratory strain amongst Toxoplasma researchers (Pfefferkorn et al., Exp. Parasitol. (1976) 39:365-376). It is highly virulent in animals and grows rapidly in culture making it ideal for obtaining large amounts of material. However, it has lost the ability to go through the complete sexual cycle in cats. Accordingly, more recent isolates, "C” and "P” strains which retain full biological function but are less virulent (Pfefferkorn et al., J. Parasitol. (1977) 63:158-159 and Ware et al., Infect. Immun. (1987) 55:778-783), were also used.
- Parasites were generally grown in vitro in monolayers of cultured human foreskin fibroblasts (HFF). Typically, using the RH strain, infected cultures were maintained by seeding uninfected monolayers at about a 1:50 dilution every 48-72 hours. This yields about 10 9 parasites from three T175 flasks of infected cultures. Parasites were harvested just as lysis occurred by passage of trypsinized cells through a syringe and removal of HFF debris by column chromatography, as described in Hoshino-Shimizu et al., J. Parasitol (1980) 66:989-991.
- ⁇ kRHg1 a library of needle-sheared genomic DNA from RH strain.
- CRHg1 a library of partially Sau3A digested RH genomic DNA inserted into the BamHI site of the cosmid vector c2XB (Bates et al., Gene (1983) 26:137146).
- HC1 . . . HC10 human sera from infants congenitally infected with T. gondii.
- HA human sera from infected human adults.
- Rp30 rabbit antisera to purified p30 (prepared by immunoadsorption to mcAb 7B8).
- RTL1 and RTL2 rabbit antisera to lysates of T. gondii RH strain tachyzoites.
- Polyclonal antisera to p30 was used to screen a cDNA library, 1RHc2.
- Several recombinants were identified on the first screen, and of these, three were initially chosen for further examination based on the strength and reproducibility of the positive signal.
- the three recombinants were compared by isolating the inserts and using each as a hybridization probe against the other and in Southern blot analyses of digested genomic DNA. From this, it became clear that the three recombinants represent different genes, implying that at least two were due to fortuitous cross-reaction with the anti-p30 sera. Sequence and Southern blot analyses confirmed their distinct coding functions.
- rabbit antisera was prepared to each fusion protein by excising the appropriate band from an acrylamide gel and injecting this into a rabbit. These sera were then used in Western blot analyses against a lysate of T. gondii. Only antisera to one clone, ⁇ Tc30.5, showed reactivity to p30. This sera also showed reactivity to no other material in the lysate. That this was indeed p30, rather than a comigrating material, is evident from the reactivity of the antisera with purified p30. The other two clones are clearly distinct genes and presumably only fortuitously cross-reactive.
- the ⁇ Tc30.5 clone was fully sequenced and used to isolate other cDNA clones which have also been sequenced. From these, a complete sequence for the p30 coding region has been derived.
- the primary translation product has a predicted Mr of 36,210 kD. The identity of this gene as that encoding p30 was confirmed by sequencing of the purified protein, which established that the mature protein results from removal of a signal peptide.
- the p30 antigen also has a probable hydrophobic signal peptide at its N-terminus, as expected for a surface antigen. It has one predicted N-glycosylation site (residue 267) consistent with prior results indicating that p30 may be a glycoprotein.
- cyanogen bromide fragments can be predicted.
- the data indicate two large fragments, one of which has a tyrosine.
- cyanogen bromide generates a single large fragment (by polyacrylamide gel electrophoresis) of the predicted size (11 kD) along with several small fragments.
- the polypeptide sequence predicts substantial hydrophobicity for the protein as a whole as previously indicated for p30 by charge-shift immunoelectrophoresis.
- the presumptive p30 gene is present in one copy per haploid genome and encodes a mRNA of 1.5 kb. Based on the signal intensity of the Northern analyses (the band is readily apparent in 0.25 hr) and abundance of cDNAs for this gene in the cDNA library (at least 20 plaques per 10,000 recombinant phage), it is an abundant message as would be expected of a protein present at about 3% of total cellular protein.
- the P and C strain p30 genes were cloned using the polymerase chain reaction and oligonucleotides based on the RH strain sequence. Sequence analysis of the cloned product was by conventional technologies.
- Toxoplasma gondii Twenty strains of Toxoplasma gondii have been analyzed with respect to the following properties: virulence, restriction-fragment length polymorphisms (RFLPs), and (for six of the twenty) antigenicity. These strains and their relative virulence are generally well known to those involved in the study of Toxoplasma but largely only with regard to the first parameter. These three parameters are discussed in more detail below.
- Virulence is measured in mice as the number of parasites (tachyzoite form) which must be injected intraperitoneally in order to achieve killing of 100% of Balb/C mice (lethal dose or LD 100 ).
- Each of the twenty strains falls into one of two categories: those which have an LD 100 of less than ten organisms (termed “virulent”) and those which have an LD 100 of over 1000 organisms (termed “avirulent").
- the bimodality of this distribution i.e., the fact that the relative virulence of the strains does not form a continuum is well known.
- the gene encoding the major surface antigen, p30 has been cloned and sequenced from three strains.
- the first such sequence was for the virulent RH strain (Burg, J. L., Perelman, D., Kasper, L. H., Ware, P. L. and Boothroyd, J. C. 1988. Molecular analysis of the gene encoding the major surface antigen of Toxoplasma gondii. J. Immun. 141:3584-3591). Subsequently, the sequence of the p30 gene from two avirulent strains (P and C) was determined as reported here.
- a mouse hybridoma has been generated that produces a monoclonal antibody specific for p30 from RH strain Toxoplasma gondii. This was generated by injecting membrane fractions from RH strain parasites into mice, harvesting their spleens, generation of hybridomas by conventional means and screening of the resulting hybridomas for those secreting an antibody specific for p30. Preparation and screening is entirely conventional now that a virulence-determining factor (the p30 antigen) is known. The initial screening step was done by first examining the reactivity of the mAb by Western blot analysis in which whole lysates of T.
- gondii strain RH were resolved on a polyacrylamide gel, transferred to nitrocellulose and incubated in the presence of the supernatant from the many hybridomas, individually.
- Those mAbs that were believed to be specific for p30 based on their ability to react with a protein of the mobility known for p30 were further characterized. Confirmation that they were specific for p30 was based on demonstration that they react with a preparation of purified, homogeneous p30 from strain RH.
- a strain is of the virulent or avirulent type can be assessed simply by determining whether it binds the 9B2 monoclonal antibody (or any antibody with the same virulence specificity) and/or whether it possesses one or other of the virulent-specific restriction sites.
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Abstract
Description
TABLE 1 ______________________________________ PCR ANALYSIS OF HUMAN ISOLATES BASED ON p30 LOCUS Isolate Location Ddel HaII Sau961 N/V ______________________________________ NON-AIDS ISOLATES: Tg51 France - + - N Tg68 USA:CA - + - N Tg96 Australia - + - N Tg132 Japan - + - N BEV UK - + - N RH-R USA(JSR) + - + V RH-88 USA(ERP) + - + V OH3 Brazil + - + V AIDS ISOLATES: HAR USA - + - N MOR USA + - + V SOL USA + - + V ______________________________________ N = nonvirulent V = virulent
TABLE 2 ______________________________________ PCR ANALYSIS OF ANIMAL ISOLATES BASED ON p30 LOCUS Isolate Host Location Ddel HaeII Sau961 N/V ______________________________________ ME49 Sheep USA:CA - + - N PLK Sheep (ME49) - + - N CEP Cat USA:NH - + - N M7741 Sheep USA:IO - + - N C56 Chicken USA:CA - + - N Tg17 Pig Japan - + - N GT1 Goat USA:MD + - + V CT1 Bovine USA:MD + - + V S11 Pig Brazil + - + V ______________________________________ N = nonvirulent V = virulent
__________________________________________________________________________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 4 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1183 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 160..1170 (D) OTHER INFORMATION: /codon.sub.-- start=160 /function="SURFACE ANTIGEN OF NON-VIRULENT T. GONDII STRAIN" /product="TYPE II P30 ANTIGEN" /gene="SAG1" /standard.sub.-- name="P30" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CAATGTGCACCTGTAGGAAGCTGTAGTCACTGCTGATTCTCGCTGTTCTCGGCAAGGGCT60 GACGACCGGAGTACAGTTTTTGTGGGCAGAGCCGCTGTGCAGCTTTCCGTTGTTCTCGGT120 TGTGTCACATGTGTCATTGTCGTGTAAACACA CGGTTGTATGTCGGTTTCGCTG174 MetSerValSerLeu 15 CACCACTTCATTATTTCTTCTGGT TTTTTGGCGAGTATGTTTCCGAAG222 HisHisPheIleIleSerSerGlyPheLeuAlaSerMetPheProLys 101520 GCAGTGAGACGCGCCGTCACGGCA GGGGTGTTTGCCGCGCCCACACTG270 AlaValArgArgAlaValThrAlaGlyValPheAlaAlaProThrLeu 253035 ATGTCGTTCTTGCGATGTGGCGCTAT GGCATCGGATCCCCCTCTTGTT318 MetSerPheLeuArgCysGlyAlaMetAlaSerAspProProLeuVal 404550 GCCAATCAAGTTGTCACCTGCCCAGATAAAA AATCGACAGCCGCGGTC366 AlaAsnGlnValValThrCysProAspLysLysSerThrAlaAlaVal 556065 ATTCTCACACCGACGGAGAACCACTTCACTCTCAAGTGC CCTAAAACA414 IleLeuThrProThrGluAsnHisPheThrLeuLysCysProLysThr 70758085 GCGCTCACAGAGCCTCCCACTCTTGCGTACTCACCC AACAGGCAAATC462 AlaLeuThrGluProProThrLeuAlaTyrSerProAsnArgGlnIle 9095100 TGCCCAGCGGGTACTACAAGTAGCTGTACATCAAA GGCTGTAACATTG510 CysProAlaGlyThrThrSerSerCysThrSerLysAlaValThrLeu 105110115 AGCTCCTTGATTCCTGAAGCAGAAGATAGCTGGTGGA CGGGGGATTCT558 SerSerLeuIleProGluAlaGluAspSerTrpTrpThrGlyAspSer 120125130 GCTAGTCTCGACACGGCAGGCATCAAACTCACAGTTCCAATC GAGAAG606 AlaSerLeuAspThrAlaGlyIleLysLeuThrValProIleGluLys 135140145 TTCCCCGTGACAACGCAGACGTTTGTGGTCGGTTGCATCAAGGGAGAC 654 PheProValThrThrGlnThrPheValValGlyCysIleLysGlyAsp 150155160165 GACGCACAGAGTTGTATGGTCACAGTGACAGTACAAGCCAGAGCCTC A702 AspAlaGlnSerCysMetValThrValThrValGlnAlaArgAlaSer 170175180 TCGGTCGTCAATAATGTCGCAAGGTGCTCCTACGGTGCAAACAGCA CT750 SerValValAsnAsnValAlaArgCysSerTyrGlyAlaAsnSerThr 185190195 CTTGGTCCTGTCAAGTTGTCTGCGGAAGGACCCACTACAATGACCCTC 798 LeuGlyProValLysLeuSerAlaGluGlyProThrThrMetThrLeu 200205210 GTGTGCGGGAAAGATGGAGTCAAAGTTCCTCAAGACAACAATCAGTAC84 6 ValCysGlyLysAspGlyValLysValProGlnAspAsnAsnGlnTyr 215220225 TGTTCCGGGACGACGCTGACTGGTTGCAACGAGAAATCGTTCAAAGAT894 CysSer GlyThrThrLeuThrGlyCysAsnGluLysSerPheLysAsp 230235240245 ATTTTGCCAAAATTAAGTGAGAACCCGTGGCAGGGTAACGCTTCGAGT942 Ile LeuProLysLeuSerGluAsnProTrpGlnGlyAsnAlaSerSer 250255260 GATAATGGTGCCACGCTAACGATCAACAAGGAAGCATTTCCAGCCGAG990 As pAsnGlyAlaThrLeuThrIleAsnLysGluAlaPheProAlaGlu 265270275 TCAAAAAGCGTCATTATTGGATGCACAGGGGGATCGCCTGAGAAGCAT1038 SerL ysSerValIleIleGlyCysThrGlyGlySerProGluLysHis 280285290 CACTGTACCGTGCAACTGGAGTTTGCCGGGGCTGCAGGGTCAGCAAAA1086 HisCysThr ValGlnLeuGluPheAlaGlyAlaAlaGlySerAlaLys 295300305 TCGTCTGCGGGAACAGCCAGTCACGTTTCCATTTTCGCCATGGTGACC1134 SerSerAlaGlyThrAla SerHisValSerIlePheAlaMetValThr 310315320325 GGACTTATTGGCTCTATCGCAGCTTGTGTCGCGTGAGTGATTACCGTTG1183 GlyLeuIleGlySe rIleAlaAlaCysValAla 330335 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1183 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (i v) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 160..1170 (D) OTHER INFORMATION: /codon.sub.-- start=160 /function="SURFACE ANTIGEN OF VIRULENT STRAIN" /product="TYPE I P30 ANTIGEN" /gene="SAG1" /standard.sub.-- name="P30" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CAATGTGCAC CTGTAGGAAGCTGTAGTCACTGCTGATTCTCACTGTTCTCGGCAAGGGCC60 GACGACCGGAGTACAGTTTTTGTGGGCATAGCCGCTGTGCAGCTTCCCGTTGTTCTCGGT120 TGTGTCACATGTGTCATTGTCGTGTAAACACACGGTTGTATGTCGGTTTCGCT G174 MetSerValSerLeu 15 CACCACTTCATTATTTCTTCTGGTTTTTTGGCGAGTATGTTTCC GAAG222 HisHisPheIleIleSerSerGlyPheLeuAlaSerMetPheProLys 101520 GCAGTGAGACGCGCCGTCACGGCAGGGGTGTTTGCCGCGCCCA CACTG270 AlaValArgArgAlaValThrAlaGlyValPheAlaAlaProThrLeu 253035 ATGTCGTTCTTGCGATGTGGCGTTATGGCATCGGATCCCCCTCTT GTT318 MetSerPheLeuArgCysGlyValMetAlaSerAspProProLeuVal 404550 GCCAATCAAGTTGTCACCTGCCCAGATAAAAAATCGACAGCCGCGGTC 366 AlaAsnGlnValValThrCysProAspLysLysSerThrAlaAlaVal 556065 ATTCTCACACCGACGGAGAACCACTTCACTCTCAAGTGCCCTAAAACA414 Ile LeuThrProThrGluAsnHisPheThrLeuLysCysProLysThr 70758085 GCGCTCACAGAGCCTCCCACTCTTGCGTACTCACCCAACAGGCAAATC462 AlaLeuThrGluProProThrLeuAlaTyrSerProAsnArgGlnIle 9095100 TGCCCAGCGGGTACTACAAGTAGCTGTACATCAAAGGCTGTAACATTG510 CysProAlaGlyThrThrSerSerCysThrSerLysAlaValThrLeu 105110115 AGCTCCTTGATTCCTGAAGCAGAAGATAGCTGGTGGACGGGGGATTCT558 S erSerLeuIleProGluAlaGluAspSerTrpTrpThrGlyAspSer 120125130 GCTAGTCTCGACACGGCAGGCATCAAACTCACAGTTCCAATCGAGAAG606 AlaSer LeuAspThrAlaGlyIleLysLeuThrValProIleGluLys 135140145 TTCCCCGTGACAACGCAGACGTTTGTGGTCGGTTGCATCAAGGGAGAC654 PheProValThrThr GlnThrPheValValGlyCysIleLysGlyAsp 150155160165 GACGCACAGAGTTGTATGGTCACGGTGACAGTACAAGCCAGAGCCTCA702 AspAlaGlnSe rCysMetValThrValThrValGlnAlaArgAlaSer 170175180 TCGGTCGTCAATAATGTCGCAAGGTGCTCCTACGGTGCAGACAGCACT750 SerValValA snAsnValAlaArgCysSerTyrGlyAlaAspSerThr 185190195 CTTGGTCCTGTCAAGTTGTCTGCGGAAGGACCCACTACAATGACCCTC798 LeuGlyProVal LysLeuSerAlaGluGlyProThrThrMetThrLeu 200205210 GTGTGCGGGAAAGATGGAGTCAAAGTTCCTCAAGACAACAATCAGTAC846 ValCysGlyLysAspGly ValLysValProGlnAspAsnAsnGlnTyr 215220225 TGTTCCGGGACGACGCTGACTGGTTGCAACGAGAAATCGTTCAAAGAT894 CysSerGlyThrThrLeuThrGlyCy sAsnGluLysSerPheLysAsp 230235240245 ATTTTGCCAAAATTAACTGAGAACCCGTGGCAGGGTAACGCTTCGAGT942 IleLeuProLysLeuThrGluA snProTrpGlnGlyAsnAlaSerSer 250255260 GATAAGGGTGCCACGCTAACGATCAAGAAGGAAGCATTTCCAGCCGAG990 AspLysGlyAlaThrLeuThr IleLysLysGluAlaPheProAlaGlu 265270275 TCAAAAAGCGTCATTATTGGATGCACAGGGGGATCGCCTGAGAAGCAT1038 SerLysSerValIleIleGlyCys ThrGlyGlySerProGluLysHis 280285290 CACTGTACCGTGAAACTGGAGTTTGCCGGGGCTGCAGGGTCAGCAAAA1086 HisCysThrValLysLeuGluPheAlaGl yAlaAlaGlySerAlaLys 295300305 TCGGCTGCGGGAACAGCCAGTCACGTTTCCATTTTTGCCATGGTGATC1134 SerAlaAlaGlyThrAlaSerHisValSerIlePheA laMetValIle 310315320325 GGACTTATTGGCTCTATCGCAGCTTGTGTCGCGTGAGTGATTACCGTTG1183 GlyLeuIleGlySerIleAlaAlaCysValAla 330335 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 336 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: (D) OTHER INFORMATION: /function="SURFACE ANTIGEN OF NON-VIRULENT T. GONDII STRAIN" /product="TYPE II P30 ANTIGEN" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: MetSerValSerLeuHisHisPheIleIleSerSerGlyPheLeuAla 151015 SerMetPh eProLysAlaValArgArgAlaValThrAlaGlyValPhe 202530 AlaAlaProThrLeuMetSerPheLeuArgCysGlyAlaMetAlaSer 35 4045 AspProProLeuValAlaAsnGlnValValThrCysProAspLysLys 505560 SerThrAlaAlaValIleLeuThrProThrGluAsnHisP heThrLeu 65707580 LysCysProLysThrAlaLeuThrGluProProThrLeuAlaTyrSer 8590 95 ProAsnArgGlnIleCysProAlaGlyThrThrSerSerCysThrSer 100105110 LysAlaValThrLeuSerSerLeuIleProGluAlaGluAspSerTrp 115120125 TrpThrGlyAspSerAlaSerLeuAspThrAlaGlyIleLysLeuThr 130135140 ValProIleGluLysPheProValThrTh rGlnThrPheValValGly 145150155160 CysIleLysGlyAspAspAlaGlnSerCysMetValThrValThrVal 165170 175 GlnAlaArgAlaSerSerValValAsnAsnValAlaArgCysSerTyr 180185190 GlyAlaAsnSerThrLeuGlyProValLysLeuSerAlaGluG lyPro 195200205 ThrThrMetThrLeuValCysGlyLysAspGlyValLysValProGln 210215220 AspAsnAsnGlnTyrCys SerGlyThrThrLeuThrGlyCysAsnGlu 225230235240 LysSerPheLysAspIleLeuProLysLeuSerGluAsnProTrpGln 245 250255 GlyAsnAlaSerSerAspAsnGlyAlaThrLeuThrIleAsnLysGlu 260265270 AlaPheProAlaGluSerLysSerValIleIl eGlyCysThrGlyGly 275280285 SerProGluLysHisHisCysThrValGlnLeuGluPheAlaGlyAla 290295300 AlaGly SerAlaLysSerSerAlaGlyThrAlaSerHisValSerIle 305310315320 PheAlaMetValThrGlyLeuIleGlySerIleAlaAlaCysValAla 325330335 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 336 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: (D) OTHER INFORMATION: /function="SURFACE ANTIGEN OF VIRULENT T. GONDII STRAIN" /product="TYPE I P30 ANTIGEN" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: MetSerValSerLeuHisHisPheIleIleSerSerGlyPheLeuAla 151015 SerMetPheProLysAlaValArg ArgAlaValThrAlaGlyValPhe 202530 AlaAlaProThrLeuMetSerPheLeuArgCysGlyValMetAlaSer 3540 45 AspProProLeuValAlaAsnGlnValValThrCysProAspLysLys 505560 SerThrAlaAlaValIleLeuThrProThrGluAsnHisPheThrLeu 65 707580 LysCysProLysThrAlaLeuThrGluProProThrLeuAlaTyrSer 859095 ProAsnArgGlnI leCysProAlaGlyThrThrSerSerCysThrSer 100105110 LysAlaValThrLeuSerSerLeuIleProGluAlaGluAspSerTrp 1151 20125 TrpThrGlyAspSerAlaSerLeuAspThrAlaGlyIleLysLeuThr 130135140 ValProIleGluLysPheProValThrThrGlnThrPheValVal Gly 145150155160 CysIleLysGlyAspAspAlaGlnSerCysMetValThrValThrVal 165170175 Gl nAlaArgAlaSerSerValValAsnAsnValAlaArgCysSerTyr 180185190 GlyAlaAspSerThrLeuGlyProValLysLeuSerAlaGluGlyPro 195 200205 ThrThrMetThrLeuValCysGlyLysAspGlyValLysValProGln 210215220 AspAsnAsnGlnTyrCysSerGlyThrThrLeuT hrGlyCysAsnGlu 225230235240 LysSerPheLysAspIleLeuProLysLeuThrGluAsnProTrpGln 245250 255 GlyAsnAlaSerSerAspLysGlyAlaThrLeuThrIleLysLysGlu 260265270 AlaPheProAlaGluSerLysSerValIleIleGlyCysThrGlyGly 275280285 SerProGluLysHisHisCysThrValLysLeuGluPheAlaGlyAla 290295300 AlaGlySerAlaLysSerAlaAl aGlyThrAlaSerHisValSerIle 305310315320 PheAlaMetValIleGlyLeuIleGlySerIleAlaAlaCysValAla 325 330335
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US20210024985A1 (en) * | 2019-07-26 | 2021-01-28 | Emory University | Devices and Methods Useful for Imaging Transient and Rare Mechanical Events in Cells |
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US6326008B1 (en) | 1989-12-08 | 2001-12-04 | Dade Behring Marburg Gmbh | Toxoplasma gondii antigens, the preparation thereof and the use thereof |
WO1998045288A1 (en) * | 1997-04-09 | 1998-10-15 | Eli Lilly And Company | Treatment or prophylaxis of prostatic cancer and benign prostatic hyperplasia with selective estrogen receptor modulators |
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US20030113822A1 (en) * | 2001-05-22 | 2003-06-19 | President And Fellows Of Harvard College For Inventors Westwood And Mitchison | Identification of anti-protozoal agents |
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CN109112113A (en) * | 2018-09-05 | 2019-01-01 | 四川迈克生物新材料技术有限公司 | Monoclonal antibody, hybridoma cell strain, kit and its application of anti-human igg |
CN109112113B (en) * | 2018-09-05 | 2023-01-10 | 四川安可瑞新材料技术有限公司 | Anti-human IgG monoclonal antibody, hybridoma cell strain, kit and application thereof |
US20210024985A1 (en) * | 2019-07-26 | 2021-01-28 | Emory University | Devices and Methods Useful for Imaging Transient and Rare Mechanical Events in Cells |
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